Composition and Method for Treating Connective Tissue Damage by Transmucosal Administration

ABSTRACT

The present invention provides a composition, and a method of use thereof for treating connective tissue damage in man and in animals, which comprises a therapeutically effective amount of chondroitin sulfate, N-acetyl D-glucosamine, and hyaluronan (hyaluronic acid). Particularly, the present invention provides a composition, and a method of use thereof, for treating connective tissue damage including, but not limited to, arthritic disease, osteoarthritis, rheumatoid arthritis, osterochondrosis dessicans, cartilage damage, joint injury, joint inflammation, joint synovitis, degenerative joint disease (DJD), post surgical DJD, traumatic injury, fracture, tendon damage, ligament damage, skeletal damage, musculoskeletal damage, fiber damage, adipose tissue damage, blood cell damage, and plasma damage. Compositions for delivery of the present invention include those for parenteral, oral, and transmucosal delivery and for direct surgical placement onto the affected tissues.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims continuation-in-part priority toco-pending U.S. application Ser. No. 11/015,137, filed on Dec. 17, 2004,and U.S. application Ser. No. 11/105,163, filed on Apr. 13, 2005, bothof which claim continuation-in-part priority to U.S. application Ser.No. 10/686,918, filed on Oct. 16, 2003, issued as U.S. Pat. No.6,979,679, which claims priority to U.S. Provisional Application Ser.No. 60/419,009, filed on Oct. 16, 2002 and U.S. Provisional ApplicationSer. No. 60/487,681, filed on Jul. 16, 2003, the entire contents ofwhich are all incorporated by reference herein.

FIELD OF INVENTION

The present invention is generally directed to compositions, and methodsof use thereof for treating connective tissue damage in man and inanimals. More particularly, the present invention provides aproteoglycan composition for use in treating, for example,osteoarthritis, rheumatoid arthritis, osteochondrosis dessicans,cartilage damage, joint inflammation, joint synovitis, joint injuries,degenerative joint disease, post surgical degenerative joint disease,traumatic injuries, fractures, tendon damage, ligament damage, skeletaldamage, musculoskeletal damage, bone damage, fiber damage, adiposetissue damage, blood cell damage, and plasma damage.

BACKGROUND OF THE INVENTION

The four primary types of vertebrate tissues are epithelial tissue,muscle tissue, nerve tissue, and connective tissue. Connective tissuesare typically involved in structure and support, and are usually derivedfrom mesoderm cells. Connective tissue is widespread in the body, and asthe name implies, it primarily serves a connecting function to bind orstrengthen organs or other tissues. It also functions inside the body todivide and compartmentalize other tissue structures.

In vertebrates, the most common type of connective tissue is looseconnective tissue. Loose connective tissue holds organs in place andattaches epithelial tissue to other underlying tissues. Loose connectivetissue is named based on the “weave” of its constituent fibers. Thereare three main component types of loose connective tissue: cotlagenousfibers, elastic fibers, and reticular fibers. Collagenous fibers aremade of collagen and consist of bundles of fibrils that are coils ofcollagen molecules. Elastic fibers are made of elastin and arestretchable. Reticular fibers join connective tissues to other tissues.Loose connective tissue also includes adipose tissue that stores fat.

Another type of connective tissue is fibrous connective tissue, which isfound in tendons and ligaments. Fibrous connective tissue is composed oflarge amounts of closely packed collagenous fibers. Cartilage is a formof fibrous connective tissue that is composed of closely packedcollageous fibers in a rubbery gelatinous substance called chondrin. Theskeletons of sharks are composed of cartilage. Cartilage also providesflexible support for certain structures in humans including the nose,trachea, cars, and articulating joints, for example.

Bone and blood are two other specialized connective tissues. Bone is atype of mineralized connective tissue that contains collagen and calciumphosphate, a mineral crystal. Calcium phosphate gives bone its firmness.Blood is also considered a type of connective tissue. Even though it hasa different function in comparison to other connective tissues it doeshave an extracellular matrix. The matrix is the plasma and erythrocytes,leukocytes and platelets are suspended in the plasma.

The connective tissues of humans and animals are constantly subjected tostresses and strains from mechanical forces and from diseases that canresult in afflictions, such as arthritis, joint inflammation stiffnessand connective tissue injuries such as tendonitis, bursitis, strained ortom ligaments and tendons and the like. Indeed, connective tissueafflictions are quite common, presently affecting millions of Americans.Further, such afflictions can be not only painful but, in their extreme,debilitating.

Arthritic diseases, characterized by pain, inflammation and stiffness ofthe joints leading to reduced range of mobility, are due to thedegradation of connective tissue (mainly cartilage) in joints. Suchdiseases particularly affect weight-bearing joints such as the hips,knees, spine, ankles and feet and those joints with frequent movementsuch as hands, arms and neck. For instance, osteoarthritis (OA) inparticular is a degenerative disease of the joint cartilage resulting innarrowing of the joint space and changes in the underlying bone(Barclay, et al., The Annals of Pharmacotherapy, (May, 1998) 32:574-79). Osteoarthritis is the most common form of arthritis and itaffects approximately one in ten people in North America. Osteoarthritisis not limited to humans, but occurs in other mammals such as horses,dogs, cats, mice and guinea pigs as well, making osteoarthritis one ofthe most common sources of chronic pain seen by veterinarians.

In humans, rheumatoid arthritis (RA) is a connective tissue disease thathas some similar symptoms to osteoarthritis. Rheumatoid arthritis isamong the most debilitating of all forms of arthritis, causing joints toache and throb and eventually become deformed. Sometimes these symptomsmake even the simplest daily activities difficult to manage.

The exact cause of rheumatoid arthritis is unknown, however, it isbelieved to be an autoimmune disease (Maini, et al., Aetiopathogenesisof Rheumatoid Arthritis. in Mechanisms and Modes of RheumatoidArthritis, (1995) Academic Press Ltd. pp. 25-46), in which the immunesystem attacks body tissues, e.g., the synovium, as if they were foreigninvaders, culminating in inflammatory and destructive responses injoints as well as other tissues. It has also been postulated thatrheumatoid arthritis is triggered by an infection, possibly a virus orbacterium in people with an inherited susceptibility. Some researchersalso believe that hormones may be involved in the development ofrheumatoid arthritis.

As with other forms of arthritis, rheumatoid arthritis involvesinflammation of the joints. In rheumatoid arthritis, white blood cells,whose usual job is to attack unwanted invaders, such as bacteria andviruses, move from the bloodstream into the synovium. Here, these bloodcells appear to play an important role in causing the synovial membraneto become inflamed (synovitis). This inflammation results in the releaseof proteins that, over months or years, cause thickening of thesynovium. These proteins can also damage cartilage, bone, tendons andligaments. Gradually, the joint loses its shape and alignment andeventually, it may be destroyed.

Under normal conditions, the body maintains the synovial joint in stateof homeostasis through a variety of complex hormonal and mechanicalfeedback mechanisms. Several types of insult or injury can upset thedelicate homeostatic balance. For example, repeated trauma or stress(slow chronic insult) to the joint during everyday use, e.g., athletictraining or performance, is often the inciting cause of jointinflammation and loss of homeostasis. Initially, such stress results inonly soft tissue inflammation in the form of synovitis or capsulitis(e.g., traumatic synovitis). Cartilage damage may or may not initiallybe present in the early stages of stress related injury or inflammation.However, the release of inflammatory mediators into the joint such asprostaglandins, cytokines, lysosomal enzymes and free radicals can leadto damage of articular cartilage and can cause cartilage degradation andleading to development of degenerative joint disease (DJD).

A second type of insult or injury, the osteochondral defect, e.g., achip fracture, is often associated with an acute mechanical failure ortraumatic injury, e.g., an acute racing or training injury, although,such a fracture can be due to secondary complications associated withchronic DJD. Under this scenario, the lesion often starts as atraumatically induced defect in the articular cartilage. This may occuras a fragmentation of the original tissue from the joint margins orother defect which compromises the surface and integrity of thearticular cartilage. Exposure of the supporting subchondral bone tosynovial fluid and the intermittent pressures of the synovial fluidgenerated by repeated joint movement (repeated stress and trauma oftraining or racing) can lead to progressive subchondral bone sclerosisand eventual dislodging of the chip or bone fragment. Left untreated,the resulting damage often becomes progressive and DJD results (see,e.g., Nixon et al., “EQUINE FRACTURE REPAIR”, W. B. Saunders Co., 1996(ISBN 0-7216-6754-6)).

Under either scenario, once compromised, the damage to articularcartilage is usually permanent. In general, once damaged, therapy isnormally directed at limiting or reducing joint inflammation, limitingthe release of inflammatory mediators, removal of the inciting cause(e.g., the chip) and replacement of synovial fluid components. Thesemeasures are combined with a period of rest to allow for healing andfibrocartilage deposition at the affected area. The long termtherapeutic objective is directed at slowing the progression ofdegenerative processes and controlling the clinical signs of DJD).Prevention is often aimed at limiting joint inflammation before damageto cartilage occurs and in providing proper nutritional support.

The treatment of connective tissue afflictions can be quite problematic.A simple decrease in the stress to which the connective tissue issubjected is often not an option, especially in the case of athletes andanimals such as race horses. Consequently, treatment is often directedat controlling the symptoms of the afflictions and not their causes,regardless of the stage of the degenerative process. Presently,steroids, such as corticosteroids and NSAIDs, are widely used for thetreatment of these ailments (Vidal, et al., Pharmocol. Res. Commun.,10:557-569 (1978)). However, drugs such as these, which inhibit thebody's own natural healing processes, may lead to further deteriorationof the connective tissue.

Connective tissue, for example articular cartilage, is naturallyequipped to attempt to repair itself by manufacturing and remodelingprodigious amounts of collagen and proteoglycans (PGs). This ongoingprocess is placed under stress when an injury occurs. In such cases, theproduction of connective tissue matrix (collagen and proteoglycans) candouble or triple over normal levels, thereby increasing the demand forthe building blocks of both collagens and proteoglycans. The buildingblocks for collagen are amino acids, especially proline, glycine andlysine. Proteoglycans are large and complex macromolecules comprisedmainly of long chains of modified sugars called glycosaminoglycans(GAGS) or mucopolysaccharides. The terms glycosaminoglycans andmucopolysaccharides are understood in the art to be interchangeable. Dueto their dense negative ion content, proteoglycans molecules are able toattract and retain water within the cartilage formation specifically forlubrication. Proteoglycans provide the unique mechanical properties forflexibility, resiliency, and resistance to and recovery undercompressive forces.

Glucosaminoglycans are polysaccharides which occur widely in the animalkingdom. Glucosaminoglycans that are present in the tissues ofvertebrate animals have mainly a linear structure which is repetition ofa disaccharide units composed of two monosaccharides. Five kinds ofglucosaminoglycans are found in the tissues and fluids of vertebrates:chondroitin sulfates, keratin sulfates, dermatan sulfates, heparinsulfates, and hyaluronic acid.

Proteoglycans and collagen are the chief structural elements of allconnective tissues. Their synthesis is essential for proper maintenanceand repair of connective tissues. In vitro, the introduction ofglucosamine, a key precusor for GAGS, has been demonstrated to increasethe synthesis of collagen and GAGs in fibroblasts. In vivo, topicalapplication of glucosamine has enhanced wound healing. Glucosamine hasalso exhibited reproducible improvement in symptoms and cartilageintegrity in humans with osteoarthritis (L. Bucei, NutritionalSupplement Advisor, July 1992)).

The major proteoglycans found in cartilage are chondroitin sulfate,dermatan sulfate, keratan sulfate and hyaluronic acid (also known ashyaluronan or HA). Heparin sulfate is also a proteoglycan, although itis not a component of articular cartilage. Newer names for proteoglycanssometime reference function of the core protein within the moleculefound in chondroitin sulfate and keratin sulfate, e.g., aggregan, alarge proteoglycan aggregates with hyaluronin, or reference location(e.g., decorin (dermatan sulfate), which decorates type I collagenfibrils), or reference primary structure, biglycan which has twoglysoaminoglycan chains. Chondrocytes are active cells within thecartilage matrix, which manufacture new collagen and proteoglycanmolecules while excreting enzymes, which aid in removal of damagedcartilage and proteoglycans.

Chondroitin sulfate is broken down into sulfate disaccharides andN-acetyl galactosamine. D-Glucuronic acid is a key substrate comprisingone half of the hyaluronan molecule, the other being N-acetylD-glucosamine. Chondroitin sulfate, as CS4 and CS6 within the body, isthought to be an essential glycosaminoglycan which binds water to thearticular cartilage matrix and is necessary for the formation ofproteoglycans.

In particular, chondroitin sulfate is a long hydrophilic chain ofrepeating sugars. This glycosaminoglycan binds to proteoglycan moleculesaiding in water and nutrient transportation within the articularcartilage. Chondroitin in its sulfate form includes galactosamine, aprimary substrate of hylauronan and a disaccharide pathway forproteoglycan synthesis secondary to the hexosamine pathways utilized forglycosaminoglycan production. Chondroitin sulfate chains comprise thespace formation of the cartilage matrix and integral parts of theproteoglycan molecule. Chondroitin stimulates the production ofproteoglycans, glycosaminoglycans, and collagen, which are the buildingblocks of healthy cartilage. Chondroitin sulfate also inhibits thesecretion of degenerative enzymes by the chondrocytes within articularcartilage. Chondroitin sulfates are non-toxic and work synergisticallywith glucosamine to hydrate and repair articular cartilage.

Hylauronan is an integral part of both synovial fluid and articularcartilage. Within the articular cartilage, hylauronan providesviscoelastic properties allowing ease of motion between opposingsurfaces and increasing compressive resistance. Within the synovium,hylauronan, as a component of synovial fluid, provides an effectivebarrier regulating the introduction of plasma components. Under normalconditions, the body will synthesize sufficient amounts of basecomponents to maintain and grow healthy articular cartilage, whilelimiting the production and release of destructive proteinases,inflammatory mediators and catabolic enzymes.

Glucosamine, as glucosamine 5-phosphate, is naturally occurring withinthe body and is a component in the biosynthesis of glycosaminoglycans,proteoglycans, hyaluronan, and collagen. Glucosamine is available inexogenous forms, glucosamine sulfate sodium, glucosamine hydrochlorideand N-acetyl D-glucosamine. N-acetyl D-glucosamine is also a derivativeof glucose obtained by chemical hydrolysis of chitin. Thispolysaccharide is readily soluble in water and extremely bioavailable.N-acetyl D-glucosamine binds to glucuronic acid as well as galactosemaking it a precursor to hyaluronic acid, keratan-sulfate andchondroitin sulfate. This unique derivative aids in proteoglycan,collagen and glycosaminoglycan production. N-acetyl D-glucosamine hasalso been shown to aid in the healing of soft tissue injury.

There have been countless therapeutic approaches for management of jointdisease, providing nutritional supplementation of metabolic precursorsto the diet to aid in the biosynthesis of proteoglycans, GAG's,hyaluronan, and collagen (see, U.S. Pat. Nos. 5,364,845 and 5,587,363).Numerous other disclosures also suggest the introduction of nutritionalsupplements as therapy for the treatment of connective tissues. Forinstance, U.S. Pat. No. 3,683,076 to Rovati et al. teaches thatglucosamine sulfates are useful to treat arthritic conditions. U.S. Pat.No. 3,697,652 to Rovati et al. discloses that N-acetyl glucosamine canbe used to treat degenerative afflictions of the joints. U.S. Pat. Nos.5,364,845, 5,587,363, 6,492,349, 6,271,213, and 6,583,123 to Hendersonet al. teach that glucosamine, chondroitin, manganese, and/orS-Adenosylmethionine (SAM) are used to protect and repair connectivetissue. U.S. Pat. No. 6,632,804 to Ekanauake teaches that ferrous ionand an ascorbate, and glucodamine derivative are useful in treatingosteoarthritis. U.S. Pat. No. 6,645,948 to Prtito et al. teaches anutritional composition for treating connective tissue including aglucosamine salt chondroitin sulfate, collagen and sodium hyaluronate.

In U.S. Pat. No. 5,840,715 to Florio, N-acetyl glucosamine sulfate,chondroitin sulfate, gamma linolenic acid ercosapentaenoic acid anddocosahexaneoic acid, and manganese aspartate are combined to treatarthritis symptoms. U.S. Pat. No. 5,916,565 to Rose et al. teaches acomposition comprised of D-glucosamine hydrochloride, chondroitinsulfate, cayenne, ginger, turmeric, yucca, Devil's Claw, nettle leaf,Black Cohosh, alfalfa, and celery seeds to repair and maintain damagedtissues in joints of vertebrates. In U.S. Pat. No. 5,922,692, Marinodiscloses that glucosamine sulfate and chondroitin sulfate can be addedto foodstuffs. Additional related art discloses pharmaceuticalcompositions and methods for the treatment of connective tissue inhumans and animals, such as U.S. Pat. Nos. 4,216,204, 4,782,046,4,808,576, 4,837,024, 5,141,928, 5,840,715, 5,442,053, and 5,929,050.

While all the above references have been described as being effectivefor their intended use, there remains a need in the art for atherapeutic composition which demonstrates enhanced effectiveness in thetreatment of connective tissues, exhibit other improved beneficialproperties, and provides even wider applications in the modes ofadministration. The present invention meets these needs at least inpart.

SUMMARY OF THE INVENTION

The present invention provides a composition which demonstrates enhancedeffectiveness in the prevention or treatment of connective tissuedamage. In one of the preferred embodiments of the invention, thecomposition comprises a therapeutically effective amount of chondroitinsulfate, glucosamine, and hyaluronan (hyaluronic acid). In the joint,for example, chondroitin sulfate acts to stimulate the production ofproteoglycans, glycosaminoglycans, and collagen, inhibits degenerativeenzymes excreted by the chondrocytes, and synoviocytes, and aids innutrient transportation within the synovial fluid. Glucosamine, e.g.,the presently preferred N-acetyl D-glucosamine, increases thesynoviocyte and chondrocyte production and subsequent availability ofendogenous hyaluronan by the direct in situ inclusion of its primesubstrates galactosamine (through chondroitin sulfate assimilation) andN-acetyl D-glucosamine. The exogenous hyaluronan acts to replacedepleted endogenous hyaluronan and to lubricate and coat healthy as wellas damaged articular tissue during the reparative process. The abovemodes of action are believed accurate, however, the claimed uses of thepresent compositions is not limited to such hypothesized mechanisms ofactivity for achieving efficacy.

In one preferred embodiment, the chondroitin sulfate comprised in thecomposition is preferably chondroitin 4-sulfate (CS4), chondroitin6-sulfate (CS6), or a mixture of both CS4 and CS6. A therapeuticallyeffective amount of chondroitin sulfate and N-acetl D-glucosamine ispreferably from between about 0.5 grams to about 1.5 grams of per unitdose, respectively, and the therapeutically effective amount ofhyaluronan is preferably from about 10 mg to about 50 mg per unit dose.

In another preferred embodiment, the compositions provided herein arepreferably in sterile solution, suspension, or other pharmaceuticallyacceptable formulations. The composition provided herein can be applieddirectly to the affected connective tissue, or preferably, is adaptedfor intra-articular and/or systemic or parenteral administration.Systemic administrations can include, but are not limited to,intramuscular, intravenous or subcutaneous injection or via directadsorption into the bloodstream via non-gastrointestinal transmucosal,e.g., sublingual administration.

In one embodiment, the compositions provided herein are adapted fordirect injection into a target connective tissue, e.g., a tendon, aligament or bone, or they may be adapted for direct application to thetarget tissue as in, for example, a gel or paste-like material toenhance and/or prolong contact with the target tissue. In anotherembodiment, the compositions of the invention are adapted fortransmucosal delivery for direct adsorption into the bloodstream such asby sublingual or other oral transmucosal delivery. It is contemplated bycertain embodiments of the invention that the trasnsmucosal delivery caninclude any mucosal tissue that provides a mucosal surface area fordirect adsorption into the blood stream and that does not subject thecompositions of the invention to digestion and/or other alteration viagastric or intestinal enzymes. The compositions can be provided asliquids or semi-solids for direct application to the desired mucosaltissue. The compositions can be formulated into any of a variety ofpresentations designed to enhance and/or prolong contact with thedesired mucosal tissue to promote adsorption into the bloodstream. Forexample, the compositions can be incorporated into a dissolvable orbiodegradable film for placement e.g., under the tongue or as an oral ornasal spray or other presentation designed to enhance and/or prolongcontact with the mucosa of the oropharnyx or other target mucosaltissue.

In yet another preferred embodiment, the composition provided herewithis attached to a sheet of material adapted for implantation onto orbetween tissues of a mammalian body. Preferably, the composition isimpregnated into a polymeric gauze-like material or coated onto agauze-like material or joined to the material by adhesion and/orcapillary action. The material onto which the composition is attachedmay be either a permanent implant or it may be biodegradable. In yetanother preferred embodiment, the composition provided herewith isattached to a bandage or other surgical materials, including, but notlimited to, surgical suture material, surgical staple, or a device suchas a buckle.

The composition provided herewith further may optionally comprise one ormore other therapeutic agents, including, but not limited to, syntheticand non-synthetic corticosteroid agents, nonsteroidal anti-inflammatorydrugs, antirheumatics, immunoregulators, immunosuppressant, articularfunction augmenters, interieukin production inhibitors, grow factor, orstem cells having therapeutic effects. Any drugs, agents, compounds,known and/or to be developed, showing any desired therapeutic effectsare within the scope of this invention. In other embodiments, theinvention may specifically exclude one or more of the above therapeuticagents.

The present invention provides a composition which demonstrates enhancedeffectiveness promoting healthy growth of connective tissue and in thetreatment of connective tissue damage. As used herein, the term“connective tissue” refers to loose, dense regular, and elasticconnective tissues. The loose connective tissues comprises constituentfibers including, but not limited to, collagenous fibers, elasticfibers, reticular fibers. The loose connective tissue also refers to anadipose tissue. The dense regular connective tissues include, but arenot limited to, tendons, ligaments, cartilage, skeleton, and otherfibrous connective tissues. The connective tissues used herein alsorefer to blood.

The composition of the present invention can be used in the preventionor treatment of connective tissue damage, which includes any primary orsecondary diseases or injuries to the connective tissues in humans oranimals. Such diseases or injuries include, but are not limited to,arthritic diseases, osteoarthritis (OA), rheumatoid arthritis (RA),osteochondrosis dessicans (OCD), cartilage damage, joint injuries, jointinflammation, joint synovitis, degenerative joint disease (DJD), postsurgical DJD, traumatic injuries, fractures, tendon damage, ligamentdamage, skeletal damage, musculoskeletal damage, bone damage, fiberdamage, adipose tissue damage, blood cell damage, and plasma damage.

The present invention further provides a method for preventing ortreating connective tissue damage in humans or in animals comprisingadministering to a man or animal in need thereof, a therapeuticallyeffective amount of a composition comprising chondroitin sulfate,glucosamine, e.g., N-acetyl D-glucosamine, and hyaluronan. In onepreferred embodiment, the composition is directly applied to theaffected connective tissues. In another preferred embodiment, thecomposition provided herein is adapted for intra-articular or systemicor parenteral administration. Systemic administrations can include, butare not limited to, intramuscular, intravenous, or subcutaneousinjection and transmucosal administration, e.g., sublingualadministration. The connective tissue damage referred to herein includesany primary or secondary diseases or injuries to the connective tissuesin humans or animals.

The present invention further provides a kit comprising one or morecontainers comprising the composition provided herein, which comprises atherapeutically effective amount of chondroitin sulfate, glucosamine,e.g., N-acetyl D-glucosamine, and hyaluronan, and instructions for useof the composition for preventing or treating connective tissue damagein man or in animals. The invention of the kit provided herein can alsoinclude, or specifically exclude, other separate containers for otherdrugs, agents, compounds having desired therapeutic effects. Examplesfor these drugs, agents and compounds include, but are not limited to,synthetic and non-synthetic corticosteroid agents, nonsteroidalanti-inflammatory drugs (NSAIDS), antirheumatics (e.g., diseasemodifying antirheumatic drugs (DMARDS) such as tissue necrosis factorblockers (TNFs)), immunoregulators, immunosuppressant, articularfunction augmenters, interleukin production inhibitors, growth factorssuch as cartilage derived morphogenic proteins (e.g., CDMP-2), bonemorphogenic proteins, and stem cells and/or progenitor cells havingtherapeutic effects.

The composition provided in the kit can be stored in any suitablecontainer, including but not limited to, syringes and vials, and invarious dosage units. Preferably, the dosages for chondroitin sulfateand the presently preferred glucoasamine, N-acetyl D-glucosamine, arefrom between about 0.5 grams to about 1.5 grams per unit dose,respectively, and the dosage for hyaluronan is from about 10 mg to about50 mg per unit dose. The chondroitin sulfate, N-acetyl D-glucosamine,and hyaluronan provided in the kit are formulated in sterile solutions,suspensions, or any pharmaceutically acceptable formulations, and storedin suitable containers, separately or in combination, in various dosageunits. Preferably, the containers in the kit containing the compositionsprovided herein are disposable.

In yet another preferred embodiment, the kit provided herein contains asheet of material adapted for implantation onto or between tissues of ahuman body and with the composition attached thereon. Preferably, thesheet of material is in the form of an impregnatable material and isdissolvable and biodegradable. Furthermore, the kit provided hereinincludes instructions indicating a method of use of the composition fortreating any primary or secondary diseases or injuries to the connectivetissues in humans or animals.

DETAILED DESCRIPTION OF THE INVENTION

Additional objects, advantages and other novel features of the inventionwill be set forth in part in the description that follows and in partwill become apparent to those skilled in the art upon examination of theforegoing or may be learned with the practice of the invention.Additionally, throughout this document, various publications and patentshave been cited, the contents of which are incorporated herein byreference in their entirety.

Set forth in greater detail below are specific details related to acomposition which demonstrates enhanced effectiveness in the treatmentof connective tissue damage. The composition provided herein comprises atherapeutically effective amount of chondroitin sulfate, a suitableglucosamine derivative, e.g., N-acetyl D-glucosamine, and a suitablehyaluronan (hyaluronic acid). It is believed, that chondroitin sulfateacts to stimulate the production of proteoglycans, glycosaminoglycans,and collagen, inhibits degenerative enzymes excreted by thechondrocytes, and synoviocytes, and aids in nutrient transportationwithin the synovial fluid. It is believed that glucosamine derivatives,e.g. N-acetyl D-glucosamine, increase the synoviocyte and chondrocyteproduction and subsequent availability of endogenous hyaluronan by thedirect in situ inclusion of its prime substrates galactosamine (throughchondroitin sulfate assimilation) and N-acetyl D-glucosamine. It isfurther believed that the exogenous hyaluronan acts to replace depletedendogenous HA and to lubricate and coat healthy as well as damagedarticular tissue during the reparative process. The examples andproposed mechanisms of action set forth herein are in no way intended tolimit the scope of the invention. Those of skill in the art will realizethat, given the teachings provided herein, many variations of thecompositions, methods of use thereof, are possible that will fall withinthe scope of the of the invention.

The compositions of the invention provide a unique mixture comprised ofthe naturally occurring glucosaminoglycans: chondroitin sulfates CS4 andCS6, a suitable hyaluronan (hyaluronic acid) and a suitable glucosaminederivative, e.g., N-acetyl D-glucosamine. It can be appreciated that,depending upon the target connective tissue, the suitable glucosaminederivative can be selected from any of the glucosamine derivativesincluding, but not limited to, glucosamine 5-phosphate, glucosaminesulfate sodium, glucosamine hydrochloride, N-acetyl D-glucosamine andmixtures thereof

Chondroitin sulfates are one important component of certain embodimentsof the compositions of the invention. In general, chondroitin sulfatesare widely found in the connective tissues of animals in two forms ofrepeating disaccharides of D-glucoronic acid and N-acetyl galactosamine:CS4 sulfate where n-acetyl galactosamine holds an ester sulfate in itsCS4 position or CS6 sulfate where the ester sulfate is in the CS6position. Both CS4 and CS6 chondroitin sulfate function in the articularmatrix as a major constituent. Chondroitin sulfates contribute to keepthe cartilage matrix's normal characteristics through the increase ofthe glucosaminoglycan pool used by the chondrocytes for proteoglycansynthesis, as well as slowing down the inflammatory process actingdirectly on the enzymes inhibiting the compliment cascade and byexhibiting anti-prostoglandin activity.

Another important component of certain embodiments of the compositionsof the invention, hyaluronan and its salt (e.g., sodium hyaluronate), isa natural constituent of connective tissues and synovial fluid composedof repeating disaccharide units each consisting of D-glucoranic acid andN-acetyl D-glucosamine. Within the joint capsule, the surface ofarticular cartilage is covered by a thin layer of sodium hyaluronate. Itspecifically interacts with cartilage proteoglycans to form a stabileaggregate. Within the synovial fluid it confers viscal elasticity aswell as lubricating properties. Hyaluronan aids in providing nourishmentand waste removal from the articular matrix. It also providesbiochemical activity to help prevent excess fibrous tissue from formingin the cartilage matrix.

In addition, N-acetyl D-glucosamine is also important to certainembodiments of the compositions of the invention, and is a key compoundfor cartilage matrix synthesis as it enhances chondrocyte synthesis ofglucosaminoglycans. N-acetyl D-glucosamine also possesses the ability toenhance synthesis of key components of synovial fluid by feeding bothreactions necessary for the production of hyaluronan as well as forproteoglycans. Therefore, by replacing specific glucosaminoglycans lostby the invasion of the diarthrodial joint during surgery and alsoproviding the key molecules to enhance and promote the restoration ofnormal hyaluronan and proteoglycan synthesis, the physician orveterinarian can be assured of the composition's capability to protectthe joint as well as to aid in the healing process.

As used herein, the chondroitin sulfate comprised in the composition ispreferably chondroitin 4-sulfate (CS4), chondroitin 6-sulfate (CS6), ora mixture of both CS4 and CS6. The therapeutically effective amount ofchondroitin sulfate and N-acetyl D-glucosamine is preferably frombetween about 0.5 grams to about 1.5 grams of per unit dose,respectively, and the therapeutically effective amount of hyaluronan ispreferably from about 10 mg to about 50 mg per unit dose. In oneembodiment, the therapeutically effective amount comprises about 1 gramof CS4 chondroitin sulfate, or about 1 gram of CS6 chondroitin sulfateor about 1 gram of a mixture of CS4 and CS6 chondroitin sulfate per unitdose. In another embodiment, the therapeutically effective amount ofchondroitin sulfate is about 1 gram of chondroitin sulfate comprised ofabout 40% CS4 chondroitin sulfate and about 60% CS6 chondroitin sulfate.

An especially preferred therapeutic amount of N-acetyl D-glucosamine isabout 1 gram of N-acetyl D-glucosamine per unit dose of the composition.Presently preferred therapeutic amounts of hyaluronan include from about10 mg to about 50 mg of hyaluronan per unit dose of the composition. Anespecially preferred therapeutic amount of hyaluronan is from about 20to about 40 mg of hyaluronan per unit dose of the composition.

It can be appreciated by one of skill in the art that the hyaluronan canbe selected from among any of a number of commercially availablesources, such as commercially available sodium hyaluronate, and caninclude alternative salts and metabolic precursors or metabolitesthereof. Likewise there are numerous commercially available sources ofN-acetyl D-glucosamine and chondroitin sulfate and various alternativesalts or metabolic precursors or metabolites thereof that are availablefor use in the compositions set forth herein.

Another presently preferred embodiment of the invention provides acomposition comprising a sterile solution or suspension comprising about1 gram of chondroitin sulfate as a mixture of about 40% CS4 and 60% CS6chondroitin sulfate; about 1 gram of N-acetyl D-glucosamine; and about20-40 mg but especially about 30 mg of hyaluronan (e.g., sodiumhyaluronate) per unit dose of the composition.

One example of a preferred embodiment of the invention comprises a 10 mlunit dose of the composition. The composition is made as follows. Onegram of chondroitin sulfate powder is admixed with one gram of N-acetylD-glucosamine powder. These powders are weighed, admixed and 2 ml of a10 mg/ml solution of sodium hyaluronate is added to the powder mixture.The resultant mixture of chondroitin sulfate, N-acetyl D-glucosamine andsodium hyaluronate is qs with approximately 10 ml of bacteriostaticwater to achieve a final volume of 10 ml. The final concentration ofchondroitin sulfate in the composition is 0.1 gram/ml or 10%. The finalconcentration of N-acetyl D-glucosamine in the composition is 0.1gram/ml or 10% and the final concentration of sodium hyaluronate in thecomposition is 0.2 ml/ml or 20%.

One presently preferred embodiment of the invention provides acomposition consisting essentially of therapeutic amounts of chondroitinsulfate, N-acetyl D-glucosamine; and hyaluronan. The composition mayspecifically exclude other therapeutic agents, such as analgesics, ormay specifically include other therapeutic agents, such asimmunosupressants or bactericides

In another embodiment the invention provides a composition comprisingtherapeutically effective amounts of chondroitin sulfate, N-acetylD-glucosamine, and hyaluronan wherein the molecular weight per unit doseof the composition is from between about 450,000 Daltons to about1,600,000 Daltons. In yet another embodiment, the invention provides acomposition which comprises therapeutically effective amounts ofchondroitin sulfate, N-acetyl D-glucosamine, and hyaluronan wherein themolecular weight per unit dose of the composition is from between about500,000 Daltons to about 1,400,000 Daltons. In yet another embodimentthe invention provides a composition which comprises therapeutic amountsof chondroitin sulfate, N-acetyl D-glucosamine, and hyaluronan whereinthe molecular weight per unit dose of the composition is from betweenabout 550,000 Daltons to about 700,000 Daltons, but is especially about600,000 Daltons.

In a preferred embodiment the invention provides a composition whichcomprises therapeutic amounts of chondroitin sulfate, N-acetylD-glucosamine, and hyaluronan wherein the molecular weight per unit doseof the composition is greater than about 450,000 Daltons. In anotherembodiment, the invention provides a composition which comprisestherapeutic amounts of chondroitin sulfate, N-acetyl D-glucosarnine, andhyaluronan wherein the molecular weight per unit dose of the compositionis greater than about 550,000 Daltons.

Preparation of the composition provided herein may be made byconventional methods. For example, to prepare the compositions of theinvention, the above-described ingredients are combined as the activeingredient in intimate admixture with or without a suitable carrieraccording to conventional compounding techniques. This carrier may takea wide variety of forms depending upon the form of preparation desiredfor administration, e.g., oral, sublingual, nasal, guttural, rectal,transdermal or parenteral.

In preparing the compositions in oral dosage form, any usualpharmaceutical medium may be employed. For oral liquid preparations(e.g., suspensions elixirs, and solutions), media containing forexample, water, oils, alcohols, flavoring agents, preservatives,coloring agents and the like may be used. Carriers such as starches,sugars, diluents, granulating agents, lubricants, binders,disintegrating agents, and the like may be used to prepare oral solids(e.g., powders, capsules, pills, caplets, tablets, microencapsulatedgranules, microtablets, coated granules and lozenges). Capsules ortablets are a preferred oral dosage form. Controlled release forms mayalso be used. Because of their ease in administration, lozenges,tablets, pills, caplets, and capsules represent the most advantageousoral dosage unit form, in which case solid pharmaceutical carriers areobviously employed. If desired, tablets may be sugar coated or entericcoated by standard techniques. The compositions of the present inventionmay be in the form of one or more of these oral dosage forms, i.e., asingle dosage may be in multiple forms.

The composition of the present invention can also be provided in abioavailability enhancing, non-gastrointestinal transmucosal deliveryform, such as for sublingual or intranasal delivery. Detailed discussionon sublingual absorption and its benefits for drug delivery are providedby Lea (Sublingual Absorption, Positive Health) and Tolson (BulkNutrition, HPBCD Basics) (the entire contents of these articles areincorporated by reference herewith). The transmucosal formulations mayinclude thickening carriers or polymers, such as in sprays, pastes, gelsor dissolvable tablets or sheets, which prolong adherence of thecomposition to oral or nasal mucosal membranes.

In one embodiment, the composition of the invention can be formulatedfor transmucosal delivery into flavored oral paste comprisingpolyethylene glycol, flavorings, and stevioside 90% power. In anotherembodiment, the composition of the present invention can be formulatedfor transmucosal delivery into a hydroxypropyl cellulose (e.g.,1%)/propylene glycol gel. In yet another embodiment, the composition ofthe present invention can be formulated for transmucosal delivery into ahydroxyethyl cellulose (e.g.,1.75%)/glycerin (e.g., 20%)/propyleneglycol (e.g., 30%) aqueous gel. In yet another embodiment, thecomposition of the present invention can be formulated into ahydroxyethyl cellulose (e.g., 2.5%) aqueous gel.

For parenteral products, the carrier will usually comprise sterilewater, although other ingredients may be included, e.g., to aidsolubility or for preservation purposes. Injectable suspensions may alsobe prepared, in which case appropriate liquid carriers, suspendingagents, and the like may be employed.

In some preferred embodiments, the composition provided herein ispreferably in sterile solution, suspension, or other pharmaceuticallyacceptable formulations. The composition provided herein can be applieddirectly to the affected connective tissue, or is adapted forintra-articular and/or systemic or parenteral administration. Systemicadministrations can include, but are not limited to, intramuscular,intravenous or subcutaneous injection. Thus, in one embodiment, thecompositions of the invention have been specially adapted forintra-articular use and/or parenteral (e.g., intravenous orintramuscular) are sterile solutions or suspensions comprised oftherapeutic amounts of chondroitin sulfate, N-acetyl D-glucosamine, andhyaluronan. In addition to the afore-mentioned active agents, it can beappreciated by one of skill in the art that the compositions of theinvention which are adapted for intra-articular use, and othertherapeutic use can also comprise preservatives, pharmaceutically activecarriers, excipients, stabilizers, buffers, antimicrobial growthinhibitors and the like, and the use of such is contemplated by theinvention.

It is also contemplated that other formulations are possible and arewithin the scope of the invention, e.g., a powdered formulation suitablefor reconstitution with a suitable injectable liquid or for addition toa pre-selected ravage fluid. In particular, it can be appreciated by oneof skill in the art that the active agents of the compositions can bestored in a freeze dried or lyophilized state for reconstitution and useat a desired time.

In yet another preferred embodiment, the composition provided herewithis attached to an impregnated bolus or a sheet of material adapted forimplantation directly onto or between connective tissues of a mammalianbody, for example to prevent the formation of post-operative adhesions,e.g., scar tissue formation. Preferably, the composition is impregnatedinto an absorptive gauze-like material or coated onto the material orjoined to the material by adhesion and/or capillary action to a mesh orgauze. The material onto which the composition is attached or absorbedmay be either a permanent implant or it may be biodegradable. In oneembodiment, the therapeutic composition is dispersed on or within amalleable material which can be shaped for insertion into diseased orexcised tissue spaces. In yet another preferred embodiment, thecomposition provided herewith is attached to a bandage or other surgicalmaterials, including but not limited to surgical suture material,surgical staple, or a device such as a buckle. Surgically implanteddevices and sheet of materials having drugs/compositions attachedthereon are disclosed in U.S. Pat. No. 6,534,693 (the entire contents ofwhich is incorporated herein by reference).

It is contemplated by the invention that the compositions providedherein demonstrate enhanced effectiveness in the treatment of anyprimary and/or secondary damages and/or injuries to any connectivetissues in humans and animals. Such diseases and/or injuries include,but are not limited to, arthritic diseases, osteoarthritis (OA),rheumatoid arthritis (RA), osteochondrosis dessicans (OCD), cartilagedamage, joint injuries, joint inflammation, joint synovitis,degenerative joint disease (DJD), post surgical DJD, traumatic injuries,fractures, tendon damage, ligament damage, skeletal damage,musculoskeletal damage, bone damage, fiber damage, adipose tissuedamage, blood cell damage, and plasma damage, and the like.

In one embodiment of the invention compositions and methods fortreatment of rheumatoid arthritis (RA) are provided. Rheumatoidarthritis is thought to be a human autoimmune disease characterized bychronic inflammation of the synovial joints and progressive erosion ofthe articular cartilage matrix. Although the etiology and pathology ofrheumatoid arthritis is not totally understood, certain parameters arealways present. Cytokines, free radicals, reactive oxygen species anddegrading enzymes are formed as a result of phagocytic activity in therheumatoid arthritis affected joint. Inflammatory mediators such as IL1,TNF, MMPS, RNOs have long been implicated as mediators of articulardamage in rheumatoid arthritis as well as low molecular weight HA. Thebiological response to the presence of these products in rheumatoidarthritis joints may explain the increase in GAG levels present as thebody's defense mechanisms become overwhelmed as the disease progresses.

While not wishing to be bound by any particular theories of activity, itis postulated herein that the body's normal response to rheumatoidarthritis may not be effective in controlling its pathology, and anendogenous increase of specific GAGs, as provided by the compositions ofthe invention, will result in a more favorable long term response totreatment of rheumatoid arthritis. The invention provides theadministration of a multiple GAG formulation as set forth herein willreduce the production and presence of the aforementioned inflammatorymediators via specific biological and chemical pathways.

Accordingly, the invention provides methods of treatment of rheumatoidarthritis comprising periodic administrations of therapeutic amounts ofthe compositions of the invention. It can be appreciated by one of skillin the art that the treatment regimen (e.g., frequency of administrationand dosage) will vary according to the history, signalment, clinicalstage and/or severity of the rheumatoid arthritis disease in aparticular subject. In certain embodiments of the invention, thecompositions of the invention may be used in conjunction with otherknown rheumatoid arthritis treatment agents such as, e.g., DMARDs, TNFblockers, IL-1Ras, immunosupressants and the like.

One preferred method provided by the invention is a first treatmentregimen comprising a pretreatment with, e.g., a suitable DMARD, TNFblocker, or other immunosuppressant in conjunction with the compositionof the invention, or as a separate pretreatment to provide a systemicshort term downregulation of the disease process, followed by a secondregimen of treatment with the compositions of the invention to normalizeand stabilize the body's response to alleviate the symptoms ofrheumatoid arthritis on a long term basis without the deleterious sideeffects of e.g., systemic immunosuppressant agents such as TNF blockers.It is contemplated that the initial treatment regimen with, e.g., asuitable DMARD, TNF blocker or other immunosuppressant can be inconjunction with therapeutic amounts of the compositions of theinvention.

In yet another embodiment of the invention the compositions set forthherein can further comprise a therapeutically effective amount of othersuitable therapeutics including, but not limited to, antibiotics. Forinstance, suitable antibiotics for use in the compositions providedherein include, but are not limited to any of the antibiotics that canbe adapted for intra-articular use, (see, e.g., “Infectious Arthritis”Alicia L. Bertone, pp. 397-409, in JOINT DISEASE IN THE HORSE”, W. B.Sanders, 1996 (ISBN 0-7216-5135-6)). As can be appreciated by one ofskill in the art, the choice of antibiotics and other suitabletherapeutics, and their therapeutically effective amounts, can dependmany factors including, but not limited to, e.g., the etiology of theinfectious organism being treated or personal preference of the treatingveterinarian or physician.

The compositions of the invention can also further comprise or excludeother therapeutic agents insofar as it is generally used as atherapeutic for connective tissue disease (e.g., tendonitis). Examplesof other such therapeutic agents include, but are not limited to,synthetic and non-synthetic corticosteroid agents, nonsteroidalanti-inflammatory drugs, antirheumatics, immunoregulators,immunosuppressant, articular function augmenters, and interleukinproduction inhibitors. Specific examples of corticosteroid agentsinclude, but are not limited to dexamethasone, hydrocortisone,triamcinolone, betamethasone, predonisolone, methylpredonisolone,halopredone, beclomethasone and the like.

Specific examples of non-steroidal anti-inflammatory agents include, butare not limited to diclofenac, indomethacin, ibuprofen, ketoprofen,aspirin, diflunisal, fulfenamic acid, floctafenine, tolfenamic acid,sulindac, fenbufen, salicylic acid, acemetacin, proglumetacin,naburnetone, protizinic acid, thiaprofen, oxaprozin, loxoprofen,alminoprofen, zaltoprofen, flurbiprofen, flurbiprofen and the like.

In one embodiment, the compositions of present invention can furthercomprise at least one pyrazolyl benzenesulfonamide compound, e.g., asset forth in U.S. Pat. No. 5,756,529 and U.S. Pat. No. 5,466,823, thecontents of which are incorporated herein by reference. In particular,the compositions of the invention can further comprise a diarylsubstituted pyrazole useful for treatment of inflammation and/or pain.It is specifically contemplated that the compositions of the inventioncan further comprise therapeutic amounts of any of the class of diarylsubstituted pyrazoles their isomers, analogs and/or metabolites. Inparticular, these compounds reduce inflammation and/or pain primarilyvia inhibition of cyclooxygenase-2 (COX-2). In a preferred embodiment ofthe invention, the compositions provided further comprise anon-steroidal agent that reduces inflammation and/or pain primarily viainhibition of cyclooxygenase-2 (COX-2) and with the substantial absenceof inhibition of cyclooxygenase-1 (COX-1). Examples of suitable diarylsubstituted pyrazoles for use in the compositions of the invention,include, but are not limited to, celecoxib, rofecoxib and the like.

Examples of other agents which may be added to the core compositions setforth herein include, axetil, piroxicam, tenoxicam, ampiroxicam,meloxicam, D-penicillamine, bucillamine, gold sodium thiomalate,auranofin, lobenzarit, salazosulfapyridine, methotrexate,cyclophosphamide, azathioprine, mizoribine, cyclosporin and the like.

In a particularly preferred embodiment, the invention also provides acomposition comprised of therapeutically effective amounts ofchondroitin sulfate, N-acetyl D-glucosamine, hyaluronan, and a suitableantioxidant or free radical scavenger. In one embodiment, thecompositions of the invention can further comprise a therapeutic amountof suitable superoxide dismutase (SOD) or other antioxidant including,but not limited to, examples set forth in U.S. Pat. No. 6,127,356 toCrapo et al., the contents of which are incorporated herein byreference.

The present invention further provides a method for treating connectivetissue damage in humans or in animals comprising administering to a manor animal in need thereof, a therapeutically effective amount of acomposition comprising chondroitin sulfate, N-acetyl D-glucosamine, andhyaluronan. In one preferred embodiment, the composition is directlyapplied to the affected connective tissues. In another preferredembodiment, the composition provided herein is adapted forintra-articular and/or systemic or parenteral administration. Systemicadministrations can include, but are not limited to, intramuscular,intravenous or subcutaneous injection. The connective tissue damagereferred herein include any primary or secondary diseases or injuries tothe connective tissues in humans and/or animals. Such diseases orinjuries include, but are not limited to, arthritic diseases,osteoarthritis (OA), rheumatoid arthritis (RA), osteochondrosisdessicans (OCD), cartilage damage, joint injuries, joint inflammation,joint synovitis, degenerative joint disease (DJD), post surgical DJD,traumatic injuries, fractures, tendon damage, ligament damage, skeletaldamage, musculoskeletal damage, bone damage, fiber damage, adiposetissue damage, blood cell damage, and plasma damage.

One skilled in the art will understand that, in a method for treatingdiseases of connective tissue, therapeutic dosage will vary according tothe specific condition being treated and the severity of the disease,etc., and, therefore, can be given in a single dose, and then repeatedas needed, or the dosage can be given incrementally in several smallerdosages. Thus, the compositions of the present invention can beformulated such that the recommended therapeutic dose is achieved by theadministration of a single dose or by the administration of severalsmaller doses. In certain methods, the injection is given systemicallyevery 3-4 days to weeks, and the intralesional injection may be a singleinjection or a series of injections at, e.g., 3-week intervals.

It is apparent to one skilled in the art that the compositions of thisinvention can be included in a commercial package together withinstructions for its use against a disease of connective tissue. Thus,the present invention further provides a kit comprising one or morecontainers comprising the composition provided herein, which comprises atherapeutically effective amount of chondroitin sulfate, N-acetylD-glucosamine, and hyaluronan, and instructions for use the compositionfor treating connective tissue damages in man or in animals. The kitprovided herein can also include other separate containers for otherdrugs, agents, compounds having desired therapeutic effects, including,but not limited to, synthetic and non-synthetic corticosteroid agents,nonsteroidal anti-inflammatory drugs, antirheumatics, immunoregulators,immunosuppressant, articular function augmenters, and interleukinproduction inhibitors.

The composition provided in the kit can be stored in any suitablecontainer, including, but not limited to, syringes and vials, and invarious dosage units. Preferably, the dosages for chondroitin sulfateand N-acetyl D-glucosamine are from between about 0.5 grams to about 1.5grams per unit dose, respectively, and the dosage for hyaluronan is fromabout 10 mg to about 50 mg per unit dose. The chondroitin sulfate,N-acetyl D-glucosamine, and hyaluronan provided in the kit areformulated in sterile solutions, suspensions, or any pharmaceuticallyacceptable formulations, and stored in suitable containers, separatelyor in combination, in various dosage units. Preferably, the containersin the kit containing the compositions provided herein are disposable.

In one preferred embodiment, the kit contains multiple preloadedsyringes. At least one syringe is preloaded with a preselected volume offrom between about 2 and about 10 ml of the compositions of the presentinvention in a sterile solution comprised of proportionate weight tovolume ratios of the composition comprised of e.g., about 1 gram ofchondroitin sulfate as a mixture of about 40% CS4 and 60% CS6chondroitin sulfate, about 1 gram of N-acetyl D-glucosamine, and about20-40 mg but especially about 30 mg of hyaluronan (e.g., sodiumhyaluronate) per unit dose of the composition. The preloaded syringevolume will of course vary depending upon the state of disease at thetarget tissue and species of animal, etc. The kit can also contain oneor more separate syringes preloaded with a desired amount of othertherapeutics well known in the art. In yet another preferred embodiment,the kit contains at least three separate containers, one containschondroitin sulfate, such as CS4 or CS6, or a mixture of 40% CS4 and 60%CS6, one contains N-acetyl D-glucosamine, and one contains hyaluronan(e.g., sodium hyaluronate). The three components are mixed in a desireddosage unit before it is administered.

In yet another preferred embodiment, the kit provided herein contains amaterial adapted for implantation onto or between tissues of a mammalianbody and with the composition attached thereon. Preferably, the materialis in the form of malleable semi-solid substrate, or a mesh orgauze-like carrier, and is dissolvable or biodegradable. Preferably, thecomposition is impregnated into the material or coated onto the materialor joined to the material by adhesion and/or capillary action, such asto a mesh, gauze, or gauze-like material. Alternatively, the compositionis attached to the material that also includes, but is not limited to, abandage, a surgical suture or staple material, and surgical device, suchas buckle, suitable for a surgical process or device.

Furthermore, the kit provided herein includes instructions indicating amethod of use of the composition for treating any primary or secondarydiseases or injuries to the connective tissues in humans or animals.Such diseases or injuries include, but are not limited to, arthriticdiseases, osteoarthritis (OA), rheumatoid arthritis (RA),osteochondrosis dessicans (OCD), cartilage damage, joint injuries, jointinflammation, joint synovitis, degenerative joint disease (DJD), postsurgical DJD, traumatic injuries, fractures, tendon damage, ligamentdamage, skeletal damage, musculoskeletal damage, bone damage, fiberdamage, adipose tissue damage, blood cell damage, and plasma damage.Instructions are normally in the form of a written material but are notlimited to such.

Having discussed the composition of the present invention, and themethod of use thereof, providing an enhanced effectiveness for thetreatment of connective tissue damages, it will be more clearlyperceived and better understood from the following specific exampleswhich are intended to provide examples of certain preferred embodimentsand not limit the present invention.

EXAMPLES Example 1

A three-year old intact mate thoroughbred racehorse, presented with anenlarged mid right front leg. The leg was sore upon palpationaccompanied by heat underlying the affected area. The subject horse hadbeen racing in graded stakes company with the injury being discoveredtwo days post race. Diagnostic ultrasound of the affected area revealeda thirty percent (30%) grade III core lesion of the superficial digitalflexor from zone 1B through zone 3A. A tendon splitting surgicalprocedure was performed using a 16 gauge needle directed into the coreof the lesion from caudal to cranial at half inch intervals. At each ofthe six tendon splitting sites 0.5 ml a composition comprised ofchondroitin sulfate, N-acetyl D-glucosamine, and hyaluronic acid(commercially available as POLYGLYCAN®, ArthroDynamic Technologies,Lexington, Ky.) was injected directly into the core lesion.

Post operative care included a two week hypertonic sweat of the affectedleg accompanied by handwalking. At thirty days post surgery the subjecthorse was turned out into a small paddock for limited exercise. At sixtydays post surgery and treatment with the composition of the invention,ultrasound examination revealed complete repair of the core lesion inzones 1B and 2A and a fifteen percent (15%) grade 1 lesion in zones 2Band 3A. At 150 days post surgery and treatment. The ultrasound of thesuperficial digital flexor tendon still revealed hyperechoic areas inzone 3A; however, overall good quality fiber structure was evidentthroughout the tendon including the zones previously containing the corelesion. At eight months post surgery and treatment, the tendonultrasounded within normal limits, except that in zone 3A there was mildenlargement with a haphazard fiber pattern on caudal edge The subjecthorse resumed training at eight months post treatment, and at ten monthsthe tendon remained unchanged with horse in full racing training.

Example 2

A four-year old castrated male thoroughbred race horse presented with achief complaint of an acute forelimb lameness of the left forelimb,grade 2 out of 5. History of the present illness included an acutelameness after galloping. Findings upon exam included increased degreeof lameness upon flexion, and palpation of upper suspensory ligamentincreased lameness two fold for several steps. To confirm the diagnosis,the suspected suspensory ligament was blocked with 6 ml of carbocainefrom the lateral edge and the horse jogged sound approximately fiveminutes later. Ultrasound examination of the upper suspensory ligamentrevealed a mild desmitis; however, no tearing of the ligamentous fiberswas found.

Treatment: The following day, the origin of the suspensory was injectedfrom the lateral edge with 2.5 ml of a composition comprised ofchondroitin sulfate, N-acetyl D-glucosamine, and hyaluronic acid(commercially available as POLYGLYCAN®, ArthroDynamic Technologies,Lexington, Ky.) using a 21 gauge needle. The subject horse was walkedfor three days post injection and then resumed training. The horseremained sound at three weeks post injection wherein the horse won anallowance race and remained sound following a successful return toracing.

Example 3

A four-year old intact male thoroughbred racehorse presented with achief complaint of an enlarged mid left front leg, sore upon palpationaccompanied by heat. History of the present illness included the subjecthorse returning lame after a grade 2 stakes win. Findings by diagnosticultrasound examination included a twenty-two percent (22%) grade II corelesion of the left front superficial digital flexor from zone 1B through2B.

Treatment consisted of a tendon splitting surgical procedure of theaffected area of the superficial digital flexor tendon using a 16 gaugeneedle directed into the core of the lesion from caudal to cranial athalf inch intervals. At each of the five tendon splitting sites 0.5 mlof a composition comprising chondroitin sulfate, N-acetyl D-glucosamine,and hyaluronic acid (commercially available as POLYGLYCAN®,ArthroDynamic Technologies, Lexington, Ky.) was injected, using a 21gauge needle such that a total of 2.5 ml of the composition of theinvention was used. Post surgical and injection treatment consisted of atwo week hypertonic sweat applied to the affected limb with the subjectanimal being limited to handwalking.

At thirty days post surgery the horse was turned out into a smallpaddock. At sixty days post surgery, follow-up ultrasound examination ofthe superficial digital flexor tendon revealed a ten percent (10%) grade1 core lesion in zones 1B through 2B. At one hundred twenty days postsurgery a follow-up ultrasound revealed complete resolution of theoriginal core lesion. At two hundred forty days post surgery, thefollow-up ultrasound examination revealed a normal structure to thetendon. The horse resumed training and has raced again at the allowancelevel finishing second place. The tendon remained within normal limitsof palpation following a successful return to racing and no furtherultrasounds examinations were conducted.

Example 4

An adult male domestic longhaired cat presented with a history ofanorexia and intermittent vomiting and diarrhea of approximately threeto four days duration. The subject animal had a previous history oftemporary intestinal obstruction due to hairball formation in the smallintestine. Physical examination revealed a thickened area of smallintestine in the left cranial abdomen. Radiographs of the abdomen showeda thickened area of small intestine and radiographic findings consistentwith neoplasia or a chronic partial obstruction. An exploratoryceliotomy was performed and a diseased area of proximal small intestineapproximately 10-12 inches in length was identified. The walls of theintestine were thickened and the functional lumen of the intestine wasreduced at the affected area. Several areas of focal necrosis andmesenteric adhesions were present. Approximately 15 inches of smallintestine were resected to remove the diseased portion and the twohealthy ends of intestine were anastamosed.

Prior to closure of the abdomen, the area of the intestinal anastamosiswas coated with approximately 4 ml of a composition comprised ofchondroitin sulfate, N-acetyl D-glucosamine, and hyaluronic acidaccording to the invention. Closure of the abdomen was routine and theanimal was placed on prophylactic antibiotics following surgery.Recovery from the surgery was uneventful and following a restricted dietand cage confinement, the animal returned to normal activity withinabout two weeks. Follow-up examination of the surgical sight revealed nosignificant findings, no post surgical abdominal adhesions were noted onphysical examination and follow-up radiographs and ultrasoundexamination were without significant findings.

The foregoing description of preferred embodiments of the invention havebeen presented for purposes of illustration and description. They arenot intended to be exhaustive or to limit the invention to the preciseform disclosed. Obvious modifications or variations are possible inlight of the above teachings. The embodiment was chosen and described toprovide the best illustration of the principles of the invention and itspractical application to thereby enable one of ordinary skill in the artto utilize the invention in various embodiments and with variousmodifications as are suited to the particular use contemplated. All suchmodifications and variations are within the scope of the invention asdetermined by the appended claims when interpreted in accordance withthe breadth to which they are fairly, legally and equitably entitled.

1. A composition for treating connective tissue damage in a mammalcomprising a therapeutically effective amount of: chondroitin sulfate,N-acetyl D-glucosamine, and hyaluronan, in the absence of a separateanalgesic agent, wherein said composition is in a formulation adaptedfor transmucosal administration.
 2. The composition of claim 1, whereinsaid therapeutically effective amount of chondroitin sulfate comprisesfrom between about 0.5 grams to about 1.5 grams of chondroitin sulfateper unit dose of said composition.
 3. The composition of claim 2,wherein said chondroitin sulfate is chondroitin 4-sulfate.
 4. Thecomposition of claim 2, wherein said chondroitin sulfate is chondroitin6-sulfate.
 5. The composition of claim 2, wherein said chondroitinsulfate is a mixture of chondroitin 4-sulfate and chondroitin 6-sulfate.6. The composition of claim 1, wherein said therapeutically effectiveamount of N-acetyl D-glucosamine is from about 0.5 grams to about 1.5grams of N-acetyl D-glucosamine per unit dose of said composition. 7.The composition of claim 1, wherein said therapeutically effectiveamount of hyaluronan is from about 10 mg to about 50 mg of hyaluronanper unit dose of said composition.
 8. The composition of claim 1, whreinsaid transmucosal administration is a sublingual administration.
 9. Thecomposition of claim 8, wherein said formulation for sublingualadministration comprises gel, paste, film, or any dissolvable polymericmaterial in which said composition is absorbed via blood vessels under atongue or a buccal mucosa.
 10. The composition of claim 1, wherein saidtransmucosal administration is a nasal administration.
 11. Thecomposition of claim 10, wherein said formulation for nasaladministration is a nasal spray or mist.
 12. The composition of claim10, wherein said formulation is flavored.
 13. The composition of claim9, wherein said formulation is flavored.
 14. The composition of claim 9,wherein said gel comprises hydroxypropyl cellulose and propylene glycol.15. The composition of claim 9, wherein said gel comprises hydroxyethylcellulose, glycerin, and propylene glycol.
 16. The composition of claim9, wherein said gel comprises hydroxyethyl cellulose.
 17. Thecomposition of claim 1, further comprising one or more therapeuticagents selected from the group consisting of a synthetic or anon-synthetic corticosteroid agent, nonsteroidal anti-inflammatoryagent, antirheumatic agent, immunoregulation agent, immunosuppressantagent, articular function augmentor agent, and interleukin productioninhibitor.
 18. A composition for treating connective tissue damage in amammal and in a formulation adapted for transmucosal administration,said composition consisting essentially of a therapeutically effectiveamount of: chondroitin sulfate, N-acetyl D-glucosamine, and hyaluronan,in the absence of a separate analgesic agent.
 19. A method for treatingconnective tissue damages in a mammal comprising transmucosallyadministering to a mammal in need thereof, a therapeutically effectiveamount of a composition comprising chondroitin sulfate, N-acetylD-glucosamine, and hyaluronan, in the absence of a separate analgesicagent.
 20. The method of claim 19, wherein said transmucosaladministration is sublingual or intranasal administration.
 21. A methodfor treating connective tissue damage in a mammal comprisingtransmucosally administering to a mammal in need thereof, atherapeutically effective amount of a composition consisting essentiallyof chondroitin sulfate, N-acetyl D-glucosamine, and hyaluronan, in theabsence of a separate analgesic agent.
 22. The method of claim 21,wherein said transmucosal administration is sublingual or intranasaladministration.